Structural aspects of deoxyhemoglobin S that control its gelation in vitro will be investigated using three chemical approaches. In the first part the influence of chemical modification of carboxyl groups of hemoglobin S on gelation will be studied. The crystal structure of deoxyhemoglobin S suggested that there are several carboxyl groups at or near the intermolecular contact regions. However, their role in the gelation process is not readily apparent. Toward this end, the carboxyl groups of hemoglobin S will be converted to amides or esters. The various derivatives of HbS will be purified and the carboxyl groups modified will be identified by peptide analysis. Influence of carboxyl groups modified on the gelation behavior of hemoglobin S will be determined. As a second approach, chemical cross-linking using cleavable bifunctional reagent imidoesters will be undertaken. Intermolecular cross-links will be introduced in the oxy- and deoxyhemoglobin S to understand the conformational transition of oxyhemoglobin S when it is deoxygenated. This approach will also be used to follow the progress of the gelation of deoxyhemoglobin S. The third part of the proposal deals with fragment complementation, a chemical approach of generating a native-like structure by the non-covalent complexation of the fragments of native protein. Native-like "interacting non-covalent fragment systems" of alpha and/or beta chains will be prepared, mixed with the native beta and alpha chains respectively to isolate alpha2-beta2 type of species to study their functional properties and gelation behavior. Possible reformation of the peptide bond in the fragment-complementing systems of alpha and beta chains will also be studied to develop a new route for the preparation of covalent analogues of HbS. Synthetic substitution of the amino residues in one or more segments of the fragment-complementing system will also be undertaken to study the contribution of various presumptive contact regions in the gelation of deoxyhemoglobin S. The last approach, i.e., "fragment complementation", is also expected to provide valuable information about the structure, dynamics and assembly of HbS in particular, and also of proteins having two or more subunits in general.